MOLECULAR AND GENETIC ANALYSIS OF FLAGELLAR DYNEINS

该项目使用分子生物学和遗传学来研究 鞭毛动力蛋白ATP酶是一种大的、多功能的 亚单位复合物，支持运动沿着细胞质和 鞭毛微管骨架 这项工作的长期目标是 以确定动力蛋白力的产生机制及其调节。 确定一级结构并预测高级结构， 催化重链亚单位，衣原体的基因组克隆 将对莱茵氏外臂动力蛋白α和β重链进行测序。 序列分析也将用于寻找核苷酸和微管 结合结构域和与其他机械化学酶的同源性。 将开发衣原体动力蛋白装配突变体的转化 作为表达克隆的动力蛋白基因的方法。 杂交种的表达 克隆的野生型和sup-1突变体β重链基因将用于 找出sup-1突变，它定义了这个重链的一个区域， 参与放射状辐条-中央微管对的运动调节 交互. 序列分析将揭示分子缺陷 负责抑制基因表型。 将使用基因组α重链和70 k中间链克隆 通过限制性片段定位编码每种蛋白质的遗传位点， 长度多态性（RFLP）分析。 先前确定的动力蛋白组装 然后，将通过一个基因检测器检测突变体与这些结构基因位点的等位性。 RFLP作图和用克隆基因转化的组合。

This project uses both molecular biology and genetics to study the structure and function of flagellar dynein ATPases, which are large, multi- subunit complexes that support motility along both cytoplasmic and flagellar microtubule frameworks. The long-term objective of this work are to determine the mechanism of dynein force generation and its regulation. To determine primary structure and predict higher-order structure in the catalytic heavy chain subunits, genomic clones for the Chlamydomonas reinhardtii outer arm dynein alpha and beta heavy chains will be sequenced. Sequence analysis will also be used to look for nucleotide and microtubule binding domains and homology with other mechanochemical enzymes. Transformation of Chlamydomonas dynein assembly mutants will be developed as a method to express cloned dynein genes. Expression of hybrids between cloned wild-type and sup-1 mutant beta heavy chain genes will be used to pinpoint the sup-1 mutation, which defines a region of this heavy chain involved in motility regulation by radial spoke-central pair microtubule interactions. Sequences analysis will reveal the molecular defect responsible for the suppressor phenotype. Genomic alpha heavy chain and 70 k intermediate chain clones will be used to map the genetic loci encoding each protein by restriction fragment length polymorphism (RFLP) analysis. Previously-identified dynein assembly mutants will then be tested for allelism to these structural gene loci by a combination of RFLP mapping and transformation with cloned genes.